ABSTRACT Alternative splicing is a post-transcriptional process that greatly expands the proteomic and regulatory complexity of metazoan genomes. Despite the rapid progress in uncovering the regulatory aspects of alternative splicing, relatively little is known about their functional consequences. Understanding the physiological relevance of alternative splicing, including its implication in human disease, is arguably one of the most important unsolved problems in molecular biology. In this proposal, we will establish an approach to systematically investigate the biological roles of alternatively spliced exons by focusing on unique activities that these exons impart to proteins. Alternative splicing plays a particularly prominent role in the nervous system, where it is required to sustain the identities and functioning of a complex array of neuronal populations. This is reflected in the growing list of complex brain disorders, including autism spectrum disorders (ASD), associated with known or suspected splicing defects. A highly conserved program of neuronal microexons, defined as 3-27 nucleotide- long exons, is misregulated in as many as a third of autism cases and recent findings indicate that deregulation of microexon splicing can cause autism-like phenotypes in mice. We have identified a set of 24 transcriptional regulators with switch-like regulation of microexons during early differentiation of human neurons. Among these, we identified a suspected transcriptional factor and a candidate histone methyltransferase, PRDM10, as an important regulator of early neurogenesis. The goal of this proposal is to test the hypothesis that a switch-like inclusion of microexons, specifically the PRDM10 e8 microexon, results in a switch in molecular activity that is critically required during neuronal development. This is highly significant since deregulation of alternative splicing of microexons can cause autistic-like behavior in mice and is linked to human neurodevelopmental disorders, including ASD. Understanding the molecular basis of microexon activity will help decipher how these tiny genetic elements regulate neuronal development. We propose to test our hypothesis in two specific aims. First, we will investigate the mechanistic basis of microexon-regulated molecular activity by inspecting the regulatory role of the e8 microexon spliced in the neural isoform of PRDM10. We will combine biochemical and genetic approaches to examine transcriptional, enzymatic, and protein-interacting properties of PRDM10 and their alteration upon microexon inclusion. Second, we will interrogate the impact of the PRDM10 e8 microexon on neuronal function, in particular synaptic signaling and activity-induced transcriptional responses. These studies will also establish a general screening platform for investigating the impact of microexon perturbation on synaptic function.